Recombinant protein comprising multiple multi-peptide sets, pharmaceutical composition comprising the recombinant protein, and method for preparing the recombinant protein

ABSTRACT

A recombinant protein comprising multiple multi-peptide sets includes first through fifth sequencing primers and first through fourth multi-peptide regions. The first multi-peptide region is between the first and the second sequencing primers. The second multi-peptide region is between the second and the third sequencing primers. The third multi-peptide region is between the third and the fourth sequencing primers. The fourth multi-peptide region is between the fourth and the fifth sequencing primers. In each of the multi-peptide regions, multiple functional peptides can be inserted, and manufacturing thereof can be done through expression of the recombinant protein, thereby significantly enhancing the concentrations of the functional peptides. With the combination of peptide having different functions, the recombinant protein product can provide more complete and more comprehensive functionality. This application also discloses a pharmaceutical composition comprising the recombinant protein and a method for preparing the recombinant protein.

BACKGROUND OF THE INVENTION 1. Technical Field

The present invention relates to a recombinant protein, and more particularly to a recombinant protein comprising multiple multi-peptide sets, a pharmaceutical composition comprising the recombinant protein and a method for preparing the recombinant protein.

2. Description of Related Art

A peptide is a molecule consisting of about 2 to 50 amino acids, with such a relatively small size, and can be easily absorbed by human bodies to collaborate with various protein molecules, thereby performing different functions. Peptides have been reported to have various functions, such as anti-inflammatory, antihypertensive, diabetes-treating, anti-microbial functions and extensively used in applications such as medicine, nutritional supplements, and cosmetics.

However, most functional peptides are single-function ones, and thus are less effective in treating and/or preventing diseases when not working with peptides having other synergistic functions. Technically speaking, manufacturing of a single peptide through artificial synthesis is costly. Particularly, the more the amino acids are in a peptide, the more difficult accurate synthesis of amino acid sequence is, not to mention massively simultaneous manufacturing of multiple peptides.

BRIEF SUMMARY OF THE INVENTION

In view of this, the objective of the present invention is to provide a recombinant protein comprising multiple multi-peptide sets, a pharmaceutical composition comprising the recombinant protein, and a method for preparing the recombinant protein, which allow multiple sets of multiple functional peptides to be inserted into one carrier protein, so as to achieve improved therapeutic effects or functional performance.

In order to achieve the foregoing objective, the present invention provides a recombinant protein comprising multiple multi-peptide sets, comprising: a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, and a fifth sequencing primer; and a first multi-peptide region, a second multi-peptide region, a third multi-peptide region and a fourth multi-peptide region. Each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region comprises at least one peptide. The first multi-peptide region is between the first sequencing primer and the second sequencing primer, the second multi-peptide region is between the second sequencing primer and the third sequencing primer, the third multi-peptide region is between the third sequencing primer and the fourth sequencing primer, the fourth multi-peptide region is between the fourth sequencing primer and the fifth sequencing primer.

In order to achieve the foregoing objective, the present invention further provides a pharmaceutical composition, which comprises the recombinant protein comprising multiple multi-peptide sets as described previously.

In order to achieve the foregoing objective, the present invention further provides a method for preparing a recombinant protein comprising multiple multi-peptide sets, which comprises the following steps: (a) providing a carrier protein, which comprises a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, and a fifth sequencing primer; (b) placing a first multi-peptide region between the first sequencing primer and the second sequencing primer, placing a second multi-peptide region between the second sequencing primer and the third sequencing primer, placing a third multi-peptide region between the third sequencing primer and the fourth sequencing primer, and placing a fourth multi-peptide region between the fourth sequencing primer and the fifth sequencing primer, thereby forming a recombinant protein, wherein each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region comprises at least one peptide respectively;

(c) transferring the post-replacement recombinant protein to a yeast expression system for fermentation; and (d) purifying the post-fermentation recombinant protein using starch so as to obtain the recombinant protein.

With provision of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, plural functional peptides can be inserted, and manufacturing thereof can be done through expression of the recombinant protein, thereby significantly enhancing the concentrations of the functional peptides. In virtue of the combination of the different kinds of functional peptides, the recombinant protein comprising multiple multi-peptide sets, the pharmaceutical composition comprising the recombinant protein, and the resulting recombinant protein product of the method for preparing the recombinant protein as disclosed in the present invention can provide more complete and more comprehensive functionality.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a sequence of a recombinant protein the comprises multiple sets of multiple sleep-associated peptides according to a first example of the present invention, with sequencing primers shown in boldface and highlighted in grey, the first through fourth multi-peptide regions underlined, and the original sequences of the carrier protein framed.

FIG. 2 shows a sequence of a recombinant protein the comprises multiple sets of multiple diabetes-associated peptides according to a second example of the present invention, with sequencing primers shown in boldface and highlighted in grey, the first through the fourth multi-peptide regions underlined, and the original sequences of the carrier protein framed.

DETAILED DESCRIPTION OF THE INVENTION

The technical contents and features of the present invention will be expounded with reference to specific embodiments and experiment examples together with the accompanying drawings.

Selection of Carrier Protein

In one embodiment of the present invention, human tyrosine hydroxylase (HTH) is used as the recombinant protein carrier for carrying proteins and inserting peptides. Its original protein sequence contains 497 amino acids (NCBI Accession No.: AAI43612.1, SEQ ID NO: 1). Therein, the DNA sequences corresponding to amino acid regions No. 1˜20, 61˜67, 96˜102, 132˜138, and 415˜497 are selected as five sequencing primers, and named the first sequencing primer, the second sequencing primer, the third sequencing primer, the fourth sequencing primer, and the fifth sequencing primer, respectively. The sequences of the sequencing primers are shown in Table 1 below. These primers are used for verifying, through sequencing, whether target peptides have been inserted successfully. In the present embodiment, the first sequencing primer comprises a sequence of SEQ ID NO: 2; the second sequencing primer comprises a sequence of SEQ ID NO: 3; the third sequencing primer comprises a sequence of SEQ ID NO: 4; the fourth sequencing primer comprises a sequence of SEQ ID NO: 5; and the fifth sequencing primer comprises a sequence of SEQ ID NO: 6. Regions between the sequencing primers, such as amino acid regions No. 21˜60, 68˜95, 103˜131, and 139˜414, are the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, where multiple peptides are inserted to replace the original sequences.

TABLE 1 Sequences of Sequencing Primer SEQ Primer ID No. Sequence NO. 1 atgcccaccc ccgacgccac cacgccacag gccaagggct tccgcagggc 2 cgtgtctgag 2 ccctcggagc ccggggaccc c 3 3 ctgtcccgag ctgtgaaggt g 4 4 gtgcgcctcg aggtgcgccg a 5 5 gaggctgcgg ccgtgcagcc ctaccaagac cagacgtacc agtcagtcta 6 cttcgtgtct gagagcttca gtgacgccaa ggacaagctc aggagctatg cctcacgcat ccagcgcccc ttctccgtga agttcgaccg tacacgctgg ccatcgacgt gctggacagc ccccaggccg tgcggcgctc cctggagggt gtccaggatg agctggacac ccttgcccat gcgctgagtg ccattggc

Design of DNA Sequence of Recombinant Protein Comprising Multiple Multi-Peptide Sets

The multiple multi-peptide sets to be inserted into the carrier protein are arranged and the final design is used for synthesizing the DNA sequence of the recombinant protein.

First, to make the arrangement, every peptide must be led and followed by a cleavage site for pepsin so that the peptides in the resulting recombinant protein can be decomposed, in the stomach, into individual peptide amino acid sequences and function as intended. To this end, the pepsin cleavage sites are located at the N terminal and the C terminal of phenylalanine. As such, every peptide is led and followed by phenylalanine (F). In addition, for preventing phenylalanine inherent in peptides from being confused with the cleavage sites for pepsin, the phenylalanine originally existing in each peptide is replaced with tryptophan (W) or tyrosine (Y). At last, in view that different peptides in each multi-peptide set could have identical amino acid sequences, for preventing errors in DNA synthesis and expression caused by repetition of DNA sequences between different sets of multi-peptide in the recombinant protein, the entropy of DNA sequences of the recombinant protein is increased. There are two approaches to increasing entropy. The first approach is about shuffling the order of peptides. Specifically, in the sequence of every set of multi-peptide, the multi-peptide are such arranged that their order is different from the multi-peptide in any other set. The second approach is about, based on an expression system of Yarrowia lipolytica, with reference to a codon usage table, using codons of the DNA sequences corresponding to individual amino acids randomly, so that different amino acid sequences composed of multiple peptides can contain the same amino acid sequence of multi-peptide, and express the same multi-peptide, yet the DNA sequence of each set of multi-peptide is different from any other set.

After the multiple multi-peptide to be inserted into the carrier protein are sequenced, the corresponding DNA sequences are sequenced according to the principle a explained previously. With reference to the codon usage table, the codons of the DNA sequences corresponding to individual amino acids are used randomly to produce DNA sequences of multiple sets of multi-peptide. Then the DNA sequences of the sets of multi-peptide are used to replace the DNA sequences corresponding to the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region of the foregoing carrier protein, i.e., the DNA sequences corresponding to amino acid regions No. 21˜60, 73˜90, 103˜131, and 144˜414, and 12 histidines are attached to the tail for protein purification. Furthermore, a HindIII cleavage site is added to the head while a KpnI cleavage site is added to follow the rear stop codon, thereby forming a recombinant protein comprising multiple multi-peptide sets and its DNA sequence. The DNA sequence of the recombinant protein such designed is finally used for synthesis.

It is to be noted that the peptides to be inserted into the carrier protein may be selected according to design needs. The insert may be a single peptide, or more than one peptide. According to the experiments conducted by the inventors, the insert could be up to 20 different kinds of peptides. The size of the DNA sequence of each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region can be between about 700 and 1200 base pairs, which will be about 200 to 400 amino acids after translation. The DNA sequence of the designed recombinant protein comprising multiple multi-peptide sets is sized about 3000 base pairs.

Transfer and Expression of Recombinant Protein Comprising Multiple Multi-Peptide Sets

The DNA sequence of the recombinant protein comprising multiple multi-peptide sets obtained in the previous step is transferred into yeast using the HindIII and KpnI cleavage sites by means of a YLEX expression kit (YEASTERN BIOTECH CO., LTD., Taiwan), with pYLSC1-5S (Pin-Cheng Yan's Master's Thesis, Development and Bioproduction of Protein Containing the Caseinophospho-peptide for Anti-osteoporosis, Department of Molecular Biology Technology, Dayeh University, 2012) constructed previously as the plasmid. Therein, the yeast selected is Yarrowia lipolytica, which is biologically safe and does not produce endotoxin. pYLSC1-5S is inserted into the 5S-rRNA genes in the chromosome of the yeast. Since there are 82˜97 5S-rRNA genes in the chromosome of the yeast that work as insertion sites, the yield of the recombinant protein can be amplified 82˜97 times.

Afterward, with the foregoing sequencing primers, DNA sequencing is performed on the yeast having the recombinant protein that comprised multiple multi-peptide sets transferred therein, so as to confirm that the DNA sequences of the inserted peptides did exist in the yeast. After the yeast is conformed as having the sequences of the multiple multi-peptide sets inserted, expression of the recombinant protein is made, and the recombinant protein is purified using starch, thereby obtaining the desired recombinant protein product comprising multiple multi-peptide sets.

EXAMPLE 1

Table 2 shows 16 functional peptides having different properties selected in Example 1 for their ability to mitigate sleep disorder, wherein the first peptide is delta sleep-inducing peptide (DSIP) for activating drowsiness; the second peptide is growth hormone-releasing hormone (GHRH) for promoting slow-wave sleep, secreting growth hormone, and inhibiting release of stress hormone, or cortisol; the third to the seventh one are galanin, neuropeptide Y, vasopressin, oxytocin, and vasoactive intestinal peptide (VIP). These are related to melatonin synthesis. The eighth through the 11^(th) ones are ghrelin, melanin-concentrating hormone (MCH), epithalon, and vilon. These are related to normal vitality and immunity implementation. The 12^(th) is soybean-protein-derived peptide (SBP) for enhancing melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2). MT1 can regular sleep, and MT2 is related to circadian rhythm. The 13^(th) through the 15^(th) are selank, casein peptide and semax. Therein, selank is antianxiety and anti-depression, casein peptide can reduce stress hormone, or cortison. Semax is a neurotrophin capable of enhancing mental and physical performance. The last, the 16^(th) is anti-allergical peptide (AAP) that helps resist all allergens.

TABLE 2 Peptides associated with treatment of sleep disorder Name Unmodified Sequence SEQ ID NO. Modified Sequence DSIP WAGGDASGE 7 — GHRH YADAIFTNSYRKVLGQLS 8 YADAIWTNSYRKVLGQL ARKLLQDIMSRQ SARKLLQDIMSRQ Galanin GWTLNSAGYLLGPHAVG 9 GWTLNSAGYLLGPHAVG NHRSFSDKNGLTS NHRSWSDKNGLTS Neuropeptide YPSKPDNPGEDAPAEDLA 10 — Y RYYSALRHYINLITRQRY Vasopressin CYFQNCPRG 11 CYW/YQNCPRG Oxytocin CYIQNCPLG 12 — VIP HSDAVFTDNYTRLRKQM 13 HSDAVWTDNYTRLRKQM AVKKYLNSILN AVKKYLNSILN Ghrelin GSSFLSPEHQRVQQRKES 14 GSSWLSPEHQRVQQRKES KKPPAKLQPR KKPPAKLQPR MCH DTMRCMVGRVYRPCWEV 15 — Epithalon AEDG 16 — Vilon KE 17 — SBP SWGEDWGEIW 18 — Casein Peptide YLGYLEQLLR 19 — Selank TKPRPGP 20 — Semax MEHFPGP 21 MEHWPGP AAP TDGVTYTNDCL 22 —

First, the peptides SEQ ID NOs: 7˜22 as shown in Table 2 were arranged in order, and phenylalanine (F) was added to the front and the back of the combination of the sequences of every two peptides. Then the phenylalanine originally in any peptide sequence was replaced with tryptophan (W) or tyrosine (Y). In the present example, in the peptide sequence of SEQ ID NO.: 8, phenylalanine at the sixth position was replaced by tryptophan (F->W); in the peptide sequence of SEQ ID NO.: 9, phenylalanine at the 22^(nd) position was replaced by tryptophan (F->W); in the peptide sequence of SEQ ID NO.: 11, phenylalanine at the third position was replaced by tryptophan or tyrosine (F->W/Y); in the peptide sequence of SEQ ID NO.: 13, phenylalanine at the sixth position was replaced by tryptophan (F->W); in the peptide sequence of SEQ ID NO.: 14, phenylalanine at the fourth position was replace by tryptophan (F->W); and in the peptide sequence of SEQ ID NO.: 21, phenylalanine at the fifth position was replaced by tryptophan (F->W). The peptide sequences after foregoing replacement are shown in the last column in Table 2, with the replacement underlined.

At last, for adjusting the overall length of the post-insertion peptide sequence, the original sequences of the carrier protein were inserted between selected peptides. In the present example, the peptide sequence having sequences arranged in the order of SEQ ID NOs.: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, with cleavage sites added and replacement done, had a total length equivalent to 264 amino acids. Twelve original sequences (DAKQAEAIMSPR, EEKEGKAVLNLL, and EAKIHHLETRPA) of the carrier protein and one phenylalanine were inserted between SEQ ID NOs.: 12 and 13 to form three different multi-peptide sequences, defined as the first multi-peptide sequence, the second multi-peptide sequence, and the third multi-peptide sequence herein. Then SEQ ID NOs.: 7, 8, 9, 10, 11, 12 were arranged in order, and with the cleavage sites added and replaced, formed a multi-peptide sequence having a length of 130 amino acids, namely the fourth multi-peptide sequence. A last, the first multi-peptide sequence was inserted to the amino acid regions Nos. 21˜60 of the carrier protein; the second multi-peptide sequence was inserted to the amino acid region Nos. 73˜90 of the carrier protein; the third multi-peptide sequence was inserted to the amino acid regions Nos. 103˜131 of the carrier protein; the fourth multi-peptide sequence was inserted to the amino acid region Nos. 144˜414, thereby forming a recombinant protein sequence (SEQ ID NO.: 23) comprising multiple sets of multiple sleep-associated peptide, as shown in FIG. 1 .

The sequence of the foregoing recombinant protein comprising multiple multi-peptide sets was increased in entropy as described above and a DNA sequence (SEQ ID NO.: 24) corresponding thereto was reversely designed. Twelve histidines were attached to its tail and HindIII and KpnI cleavage sites were added to the front and rear ends, respectively. Then the YLEX expression kit was used to insert the recombinant protein DNA sequence to the pYLSC1-5S plasmid, which was afterward transferred to yeast for expression, thereby obtaining a powdered recombinant protein product comprising multiple sets of multi-peptide as listed in Table 2.

Testing of Functionality in Mitigating Sleep Disorder

Referring to Table 3, a group of 30 volunteers was administered with the powdered recombinant protein prepared in the manner described previously for 45 days as the treatment group. The usage and dosage were: one pack (about 2 grams) before dinner (about 6 o'clock), after dinner (between 8 and 9), and before sleep, respectively. A group of additional 20 volunteers received no treatment was provided as the control group. A group of further 25 volunteers was given a single peptide (casein peptide, SEQ ID NO.: 21) as the comparison group. At the beginning of the experiment, in each of the treatment, control, and comparison groups, 3 to 4 subjects reported poor sleep quality on Day 0, wherein 4 subjects were confirmed as having poor sleep quality after interview. During Day 2 to Day 10, only 1 to 2 subjects in the treatment group still reported poor sleep quality and the number decreased to 1 to 1.5 after Day 15. Therein, only 1 subject was confirmed as having poor sleep quality after Day 2. In the control group, 1 to 2 subjects remaining complaining of having poor sleep quality until Day 45, with 1.5 confirmed so after interview. In the comparison group, the number of subjects reporting poor sleep quality had not decreased to 1˜1.5 until Day 30, and after Day 15 there was only 1 subject confirmed as having poor sleep quality. By comparison, the treatment group performed faster improvement in sleep quality.

TABLE 3 Results of sleep quality test Day 0 Day 2 Day 5 Day 10 Day 15 Day 30 Day 45 Combined 4  1**   1***   1*** 1*** 1*** 1*** Peptide Protein (n = 30) (3-4) (1-2) (1-2) (1-2)  (1-1.5) (1-1.5) (1-1.5) No 4 4 3 3 3   2   1.5   Treatment (n = 20) (3-4) (3-4) (2-3) (2-3) (1-2) (1-2)  (1-2)  Single 4 4 3 2 1*** 1*** 1*** Peptide (n = 25) (3-4) (3-4) (2-3) (1.5-2)  (1-2) (1-1.5) (1-1.5) Wilcoxson rank test: **P < 0.01 ***P < 0.005(Vs DO) alpha level is adjusted by Bonferroni inequality calculation IQR: interquartile range

Referring to Table 4 below, the subjects were inquired for having delayed sleep or not during the test of sleep quality. In summary, the treatment group (administered with the recombinant protein product), the control group, and the comparison group (administered with a single peptide casein peptide, SEQ ID NO.: 21) each had 1 to 4 subjects reporting delayed sleep at Day 0, with 4 of them confirmed after interview. The number decreased to 1˜2 in the treatment group at after Day 2, and further decreased to 1˜1.5 after Day 15. Therein, only one subject was confirmed as having delayed sleep after Day 2. In the control group, 1 to 2 subjects remaining complaining of having delayed sleep until Day 45, with 1.5 confirmed so after interview. In the comparison group (administered with a single peptide casein peptide), 1˜subjects still reported delayed sleep after Day 10, and only one confirmed as having delayed sleep after Day 15. By comparison, the treatment group performed faster improvement in delayed sleep.

TABLE 4 Results of delayed sleep test Day 0 Day 2 Day 5 Day 10 Day 15 Day 30 Day 45 Combined 4  1**   1***   1*** 1*** 1*** 1*** Peptide Protein (n = 25) (1-4) (1-2) (1-2) (1-2) (1-1.5)  (1-1.5)  (1-1.5) No 4 4 3 3 3   2   1.5   Treatment (n = 20) (1-4) (1.5-4)  (1-4) (1-4) (1-3) (1-2) (1-2) Single 4 4 3 2 1*** 1*** 1*** Peptide (n = 25) (2-4) (2-4) (2-3) (1-2) (1-2) (1-2) (1-2) Wilcoxson rank test: **P < 0.01 ***P < 0.005(Vs DO) alpha level is adjusted by Bonferroni inequality calculation IQR: interquartile range

EXAMPLE 2

Table 5 shows 18 functional peptides having different properties selected in Example 2 for their ability to decrease plasma glucose, wherein the first to the fourth (FOL-005, FOL-014, FOL-015, FOL-047) are diabetes-treating peptides; the fifth and the sixth are human amylin (hAMY) and slim peptide PPY, for preventing diabetes and slimming, respectively; the seventh is the most potent DDP-4 inhibitor peptide, serving to inhibiting dipeptidyl peptidase 4 (DDP-4), thereby holding plasma glucose and blood pressure stable; the eighth, the ninth, and the 10^(th) peptides are inducing peptides of insulin, capable of promoting secretion of insulin, increasing the feeling of fullness, and reducing secretion of glucagon; the 11^(th) peptide is a peptide that inhibits glucose transporter 2 (GLUT2), serving to inhibiting absorption of glucose by intestinal cells, thereby reducing glucose in the blood; the 12^(th) and the 14^(th) peptides are peptides that inhibit sodium-dependent glucose cotransporters 1 (SGLT1), for decreasing high filtration of nephrocytes, keeping blood pressure and body weight constant, while reducing plasma glucose without the risk of inducing hypoglycemia: the 13^(th) peptide is a peptide inhibitor of amylin aggregation, capable of freeing monomer amylin and thereby lowering plasma glucose; the 15^(th) is insulin mimetic peptide S519, functioning like insulin in terms of lowering plasma glucose; the 16^(th) is RG33, which is an ApoA1-derived peptide tolerating glucose and preventing vascular sclerosis; the 17^(th) is an ApoA1 mimetic peptide, serving to inhibit inflammation of liver and generation of glucose and fat, and treat insulin resistance; and the last, 18^(th) is a blood glucose lowering peptide for lowering plasma glucose.

TABLE 5 Peptides associated with Lowering plasma glucose Name Unmodified Sequence SEQ ID NO. Modified Sequence FOL-005 VDTYDGDISVVYGLR 25 — FOL-014 KPLAEIDSIELSYGIK 26 — FOL-015 LDGLVRAYDNISPVG 27 — FOL-047 KPLAGIDSIGLSYGIK 28 — hAMY KCNTATCATQRLANFLV 29 KCNTATCATQRLANYLV HSSNNFGAILSSTNVGSNT HSSNNLGAILSSTNVGSNT Y Y Slim IKPEAPGEDASPEELNRYY 30 — Peptide PPY ASLRHYLNLVTRQRY Most Potent IPI 31 — DDP-4 Inhibitor Peptide Inducing HAEGTFTSDVSSYLEGQA 32 HAEGTWTSDVSSYLEGQ Peptide of AKEFIAWLVKGR AAKEWIAWLVKGR Insulin -1 Inducing LRSELAAWSR 33 — Peptide of Insulin -2 Inducing KLPGY 34 — Peptide of Insulin-3 GLUT2 ATNPLF 35 ATNPLW Inhibitor Peptide SGLT1 LSVSVL 36 — Inhibitor Peptide Peptide RGANFLVHGR 37 RGANWLVHGR Inhibitor of Amylin Aggregation SGLT1 DKLTTREIEQVELLKRIYD 38 — Inhibitor KLT Peptide Insulin SLEEEWAQVECEVYGRG 39 SLEEEWAQVECEVYGRG Mimetic CPSGSLDESFYDWFERQL CPSGSLDESWYDWWERQ Peptide G LG S519 RG33 ApoA1- PALEDLRQGLLPVLESFK 40 PALEDLRQGLLPVLESWK derived VSFLSALEEYTKKLN VSWLSALEEYTKKLN Peptide ApoA1 FAEKFKEAVKDYFAKFW 41 WAEKWKEAVKDYWAK Mimetic D WWD Peptide (4F) Plasma GHPYYSIKKS 42 — glucose Lowering Peptide

The peptides SEQ ID NOs: 25˜42 as shown in Table 5 were arranged in order, and phenylalanine (F) was added to the front and the back of the combination of the sequences of every two peptides. Then the phenylalanine originally in any peptide sequence was replaced with tryptophan (W) or tyrosine (Y). In the present example, phenylalanine at the 15^(th) position in the peptide sequence of SEQ ID NO.: 29 was replaced by tryptophan (F->W); phenylalanine at the 23^(rd) position was replaced by leucine (L) (F->L); phenylalanine at each of the 6^(th) and the 22^(nd) positions in the peptide sequence of SEQ ID NO.: 32 was replaced by tryptophan (F->W); phenylalanine at the 6^(th) position in SEQ ID NO.: 35 peptide sequence was replaced by tryptophan (F->W); phenylalanine at the 5^(th) position in the peptide sequence of SEQ ID NO.: 37 was replaced by tryptophan (F->W); phenylalanine at each of the 27^(th) and the 31^(st) positions in the peptide sequence of SEQ ID NO.: 39 was replaced by tryptophan (F->W); phenylalanine at each of the 17^(th) and the 21^(st) positions in the peptide sequence of SEQ ID NO.: 40 was replaced by tryptophan (F->W); and phenylalanine at each of the 1^(st), 5^(th), 13^(th) and 16^(th) positions in the peptide sequence of SEQ ID NO.: 41 was replaced by tryptophan (F->W). The peptide sequences after foregoing replacement are shown in the last column in Table 5, with the replacement underlined.

At last, for adjusting the overall length of the post-insertion peptide sequence, the original sequences of the carrier protein were inserted between selected peptides. In the present example, the peptide sequence having sequences arranged in the order of SEQ ID NOs.: 25, 26, 27, 28, 29, 41, 30, 31, 32, 33, 34, 35, 36, 42, with cleavage sites added and replacement done, had a total length equivalent to 236 amino acids. Twelve original sequences (DAKQAEAIMSPR) of the carrier protein and one phenylalanine were inserted between SEQ ID NOs.: 41 and 30 to form a multi-peptide sequence having a full length of 249 amino acids, which is defined as the first multi-peptide sequence herein. Then the peptide sequence having sequences arranged in the order of SEQ ID NOs.: 37, 38, 39, 40, 25, 26, 27, 28, 29, 41, with cleavage sites added and replacement done, had a total length equivalent to 229 amino acids. Twelve original sequences (EEKEGKAVLNLL) of the carrier protein and one phenylalanine were inserted between SEQ ID NOs.: 40 and 25 to form a multi-peptide sequence having a full length of 242 amino acids, which is defined as the second multi-peptide sequence herein. Then, the peptide sequence having sequences arranged in the order of SEQ ID NOs.: 30, 31, 32, 33, 34, 35, 36, 42, 37, 38, 39, 40, with cleavage sites added and replacement done, had a total length equivalent to 218 amino acids. Twelve original sequences (EAKIHHLETRPA) of the carrier protein and one phenylalanine were inserted between SEQ ID NOs.: 42 and 37 to form a multi-peptide sequence having a full length of 231 amino acids, which is defined as the third multi-peptide sequence herein. At last, the peptide sequence having sequences arranged in the order of SEQ ID NOs.: 25, 26, 27, 28, 29, 41, 30, 31, 32, 33, 34, 35, 36, 42, with cleavage sites added and replacement done, had a total length equivalent to 236 amino acids. Twelve original sequences (DAKQAEAIMSPR) of the carrier protein and one phenylalanine were inserted between SEQ ID NOs.: 41 and 30 to form a multi-peptide sequence having a full length of 249 amino acids, which is defined as the fourth multi-peptide sequence herein.

The first multi-peptide sequence was inserted to the amino acid regions Nos. 21˜60 of the carrier protein; the second multi-peptide sequence was inserted to the amino acid regions Nos. 73˜90 of the carrier protein; the third multi-peptide sequence was inserted to the amino acid regions Nos. 103˜131 of the carrier protein; the fourth multi-peptide sequence was inserted to the amino acid regions Nos. 144˜414, thereby forming a recombinant protein sequence (SEQ ID NO.: 43) comprising multiple sets of diabetes-associated multi-peptide, as shown in FIG. 2 .

The sequence of the foregoing recombinant protein comprising multiple multi-peptide sets was increased in entropy as described above and a DNA sequence (SEQ ID NO.: 44) corresponding thereto was reversely designed. Twelve histidines were attached to its tail and HindIII and KpnI cleavage sites were added to the front and rear ends, respectively. Then the YLEX expression kit was used to insert the recombinant protein DNA sequence to the pYLSC1-5S plasmid, which was afterward transferred to yeast for expression, thereby obtaining a powdered recombinant protein product comprising multiple sets of peptides as listed in Table 5.

Testing of Functionality in Controlling Plasma Glucose for Diabetes Treatment

Referring to Table 6 below, a group of 20 volunteers with T1 diabetes and 25 volunteers with T2 diabetes was administered with the powdered recombinant protein prepared in the manner described previously for 30 days as the treatment group. The usage and dosage were: one pack (about 2 grams) before breakfast, lunch, and dinner, respectively, and before sleep. A group of additional 30 volunteer (with wither T1 or T2 diabetes) was administered with a single peptide (a plasma glucose lowering peptide, SEQ ID No.: 43) as the comparison group. From records of fasting plasma glucose measured before breakfast taken before and after the administration it was found that in the treatment group, the volunteer with both T1 and T2 diabetes had decreases in plasma glucose of 190.1 mg/dl and 196 mg/dl, respectively, after the administration of the recombinant protein, while the volunteers in the comparison group only exhibited a decrease of 76 mg/dl. By comparison, the treatment group showed better ability to control plasma glucose.

TABLE 6 Results of fasting plasma glucose test Before After Administration Administration Decrease (A) Treatment Group T1D Volunteers 290.1 ± 5.7 mg/dl 100 ± 3.4 mg/dl 190.1 ± 3.3 T2D Volunteers 295.2 ± 3.2 mg/dl 99.2 ± 4.1 mg/dl   196 ± 2.7 (B) Comparison Group T1D or T2D  196 ± 7.1 mg/dl 120 ± 3.7 mg/dl   76 ± 3.1 Volunteers

To sum up, the present invention uses HTH as the carrier protein, and defines the first sequencing primer, the second sequencing primer, the third sequencing primer, the fourth sequencing primer and the fifth sequencing primer so that the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region can be inserted therebetween. No matter which multi-peptide is inserted between two sequencing primers in the carrier protein, successful insertion can be verified in the subsequent sequencing process using the same primer. This eliminates the need of design any additional primer. Moreover, with the use of multiple multi-peptide sets, peptides having certain functions or effects can be placed into a single recombinant protein for manufacturing. For people needing treatment or mitigation of specific diseases or improvements in physiological functions, multiple peptides can be delivered in a designed order, so as to achieve composite, compound-like effects. According to Examples 1 and 2, compared to no administration or administration of a single peptide, administration of the disclosed recombinant protein provides significant effects in treating particular diseases or physiological disorders.

For medical applications, the present invention may be formulated as cream, ointment, gel, pigmentum, paste, oil, softener, liposome, nanoparticles, toning lotion, mouth wash, shampoo, emulsion, spray, suppository, capsules, tablets, powder, syrup, pellets, solution, suspension, patches, or occlusive dressing.

When used in cosmetic, hair-growth or non-edible products, the recombinant protein of the present invention may be hydrolyzed in pepsin to form a liquid peptide formulation or added with proper excipients or food additives to add value of the final products and provide enhanced economic benefits.

The present invention has been described with reference to the preferred embodiments and it is understood that the embodiments are not intended to limit the scope of the present invention. Moreover, as the contents disclosed herein should be readily understood and can be implemented by a person skilled in the art, all equivalent changes or modifications which do not depart from the concept of the present invention should be encompassed by the appended claims. 

What is claimed is:
 1. A recombinant protein comprising multiple multi-peptide sets, comprising: a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, and a fifth sequencing primer; and a first multi-peptide region, a second multi-peptide region, a third multi-peptide region, and a fourth multi-peptide region, wherein each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region comprises at least one peptide; wherein the first multi-peptide region is between the first sequencing primer and the second sequencing primer; the second multi-peptide region is between the second sequencing primer and the third sequencing primer; the third multi-peptide region is between the third sequencing primer and the fourth sequencing primer; and the fourth multi-peptide region is between the fourth sequencing primer and the fifth sequencing primer.
 2. The recombinant protein of claim 1, wherein the first sequencing primer comprises a sequence of SEQ ID NO.: 2; the second sequencing primer comprises a sequence of SEQ ID NO.: 3; the third sequencing primer comprises a sequence of SEQ ID NO.: 4; the fourth sequencing primer comprises a sequence of SEQ ID NO.: 5; and the fifth sequencing primer comprises a sequence of SEQ ID NO.:
 6. 3. The recombinant protein of claim 1, wherein in each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide comprises phenylalanine at each of its two ends.
 4. The recombinant protein of claim 1, wherein in the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide is one or more than one selected from a peptide group consisting of: SEQ ID NO.: 7-22.
 5. The recombinant protein of claim 4, wherein phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 8 is replaced by tryptophan; phenylalanine at the 22^(nd) position in a peptide sequence SEQ ID NO.: 9 is replaced by tryptophan; phenylalanine at the 3^(rd) position in a peptide sequence SEQ ID NO.: 11 is replaced by tryptophan or tyrosine; phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 13 is replaced by tryptophan; phenylalanine at the 4^(th) positioning in a peptide sequence SEQ ID NO.: 14 is replaced by tryptophan; and phenylalanine at the 5^(th) position in a peptide sequence SEQ ID NO.: 21 peptide sequence is replaced by tryptophan.
 6. The recombinant protein of claim 5, wherein the recombinant protein comprises a DNA sequence of SEQ ID NO.:
 24. 7. The recombinant protein of claim 1, wherein in the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide is one or more than one selected from a peptide group consisting of: SEQ ID NOs: 25˜42.
 8. The recombinant protein of claim 6, wherein phenylalanine at the 15^(th) position in a peptide sequence SEQ ID NO.: 29 is replaced by tryptophan, and phenylalanine at the 23^(rd) position is replaced by leucine; phenylalanine at each of the 6^(th) and the 22^(nd) positions in a peptide sequence SEQ ID NO.: 32 is replaced by tryptophan; phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 35 is replaced by tryptophan; phenylalanine at the 5^(th) position in a peptide sequence of SEQ ID NO.: 37 is replaced by tryptophan; phenylalanine at each of the 27^(th) and 31^(st) positions in a peptide sequence SEQ ID NO.: 39 is replaced by tryptophan; phenylalanine at each of the 17^(th) and 21^(st) position in a peptide sequence SEQ ID NO.: 40 is replaced by tryptophan; and phenylalanine at each of the 1^(st), 5^(th), 13^(th), and 16^(th) positions in a peptide sequence SEQ ID NO.: 41 is replaced by tryptophan.
 9. The recombinant protein of claim 8, wherein the recombinant protein comprises a DNA sequence of SEQ ID NO.:
 44. 10. A pharmaceutical composition, comprising the recombinant protein of claim
 1. 11. A method for preparing a recombinant protein comprising multiple multi-peptide sets, which comprises steps of: (a) providing a carrier protein, which comprises a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, and a fifth sequencing primer; (b) placing a first multi-peptide region between the first sequencing primer and the second sequencing primer, placing a second multi-peptide region between the second sequencing primer and the third sequencing primer, placing a third multi-peptide region between the third sequencing primer and the fourth sequencing primer, and placing a fourth multi-peptide region between the fourth sequencing primer and the fifth sequencing primer, thereby forming a recombinant protein, wherein each of the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region comprises at least one peptide; (c) transferring the post-replacement recombinant protein to a yeast expression system for fermentation; and (d) purifying the post-fermentation recombinant protein using starch so as to obtain the recombinant protein.
 12. The method of claim 11, wherein the first sequencing primer comprises a sequence of SEQ ID NO.: 2; the second sequencing primer comprises a sequence of SEQ ID NO.: 3; the third sequencing primer comprises a sequence of SEQ ID NO.: 4; the fourth sequencing primer comprises a sequence of SEQ ID NO.: 5; and the fifth sequencing primer comprises a sequence of SEQ ID NO.:
 6. 13. The method of claim 11, wherein in the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide comprises phenylalanine at each of its two ends.
 14. The method of claim 11, wherein in the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide is one or more than one selected from a peptide group consisting of: SEQ ID NO.: 7˜22.
 15. The method of claim 14, wherein phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 8 is replaced by tryptophan; phenylalanine at the 22^(nd) position in a peptide sequence SEQ ID NO.: 9 is replaced by tryptophan; phenylalanine at the 3^(rd) position in a peptide sequence SEQ ID NO.: 11 is replaced by tryptophan or tyrosine; phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 13 is replaced by tryptophan; phenylalanine at the 4^(th) positioning in a peptide sequence SEQ ID NO.: 14 is replaced by tryptophan; and phenylalanine at the 5^(th) position in a peptide sequence SEQ ID NO.: 21 peptide sequence is replaced by tryptophan.
 16. The method of claim 11, wherein the recombinant protein comprises a DNA sequence of SEQ ID NO.:
 24. 17. The method of claim 11, wherein in the first multi-peptide region, the second multi-peptide region, the third multi-peptide region, and the fourth multi-peptide region, each said peptide is one or more than one selected from a peptide group consisting of: SEQ ID NO.: 25˜42.
 18. The method of claim 17, wherein phenylalanine at the 15^(th) position in a peptide sequence SEQ ID NO.: 29 is replaced by tryptophan, and phenylalanine at the 23^(rd) position is replaced by leucine; phenylalanine at each of the 6^(th) and the 22^(nd) positions in a peptide sequence SEQ ID NO.: 32 is replaced by tryptophan; phenylalanine at the 6^(th) position in a peptide sequence SEQ ID NO.: 35 is replaced by tryptophan; phenylalanine at the 5^(th) position in a peptide sequence of SEQ ID NO.: 37 is replaced by tryptophan; phenylalanine at each of the 27^(th) and 31^(st) positions in a peptide sequence SEQ ID NO.: 39 is replaced by tryptophan; phenylalanine at each of the 17^(th) and 21^(st) position in a peptide sequence SEQ ID NO.: 40 is replaced by tryptophan; and phenylalanine at each of the 1^(st), 5^(th), 13^(th), and 16^(th) positions in a peptide sequence SEQ ID NO.: 41 is replaced by tryptophan.
 19. The method of claim 11, wherein the recombinant protein comprises a DNA sequence of SEQ ID NO.:
 44. 